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Keywords:

  • antisense oligonucleotides;
  • ODC;
  • atelocollagen;
  • human cancer;
  • growth suppression

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

Substantial evidence supports a direct role of ornithine decarboxylase (ODC) in the development and maintenance of human tumors. Although antisense oligonucleotide therapy targeting various genes are useful for cancer treatment, 1 of the major limitations is the problem of delivery. A novel antisense oligonucleotide delivery method is described that allows prolonged sustainment and release of ODC antisense oligonucleotides in vivo using atelocollagen.

METHODS

The effect of ODC antisense oligonucleotides in the atelocollagen on cell growth of gastrointestinal cancer (MKN 45 and COLO201) and rhabdomyosarcoma (RD) was studied in vitro using a cell-counting method with a hemocytometer. In vivo, the effect of intratumoral, intramuscular, and intraperitoneal single administration of ODC antisense oligonucleotides in the atelocollagen on tumor growth of MKN45, COLO201, and RD cells was studied. ODC activity and polyamine contents were measured.

RESULTS

In vitro, ODC antisense oligonucleotides in the atelocollagen remarkably suppressed MKN45, COLO201, and RD cell growth. A single administration of antisense oligonucleotides in the atelocollagen via 3 routes remarkably suppressed the growth of MKN45, COLO201, and RD tumor over a period of 35–42 days.

CONCLUSIONS

As various human cancers significantly express ODC, the results strongly suggest that this new antisense method may be of considerable value for treatment of human cancers. Cancer 2007;109:993–1002. © 2007 American Cancer Society.

Polyamines, putrescine, spermidine, and spermine are essential for cell growth and differentiation.1–5 The metabolism of polyamines begins with the decarboxylation of ornithine to form putrescine regulated by ornithine decarboxylase (ODC).1–5 Increased ODC activity and polyamine levels in various human tumors have been documented.1–10 Several groups including ours reported that ODC overexpression in mouse fibroblasts induced cell transformation,11–14 and the ODC gene is now considered a protooncogene. Antisense oligonucleotides including small interfering RNA, which act by specifically hybridizing with complementary RNA sequences, can inhibit protein expression.15–18 Modified analogs, particularly phosphorothioate, have been used to specifically inhibit gene expression both in vitro and in vivo.19–22 Recent clinical studies have tested antisense compounds directed against cancer-related genes such as p53, bcl-2, c-raf, H-ras, protein kinase C-alpha, and protein kinase A.23, 24 Inhibition of target gene expression has been modest and clinical activity has not been remarkable. Continuous infusion may be necessary to achieve sufficient and sustained delivery of antisense oligonucleotides to the tumor.

Recently, the authors of the present study reported that HST-1/FGF-4 plasmid DNA embedded in the atelocollagen was protected from nuclease attack and sustained in peripheral blood for as long as 40 days after injection of plasmid DNA embedded in the atelocollagen in vivo.25 We next reported that a phosphorothioate antisense oligodeoxynucleotides of midkine embedded in atelocollagen suppressed CMT-93 mouse rectal carcinoma cell growth.26 In this study we extended these studies for elucidating the potential of antisense phosphorothioate oligonucleotides (ODNs) embedded in the atelocollagen, targeted against ODC on the growth of human gastrointestinal tumor and rhabdomyosarcoma xenograft inoculated in nude mice. Here we demonstrate that a single administration of ODC antisense ODNs embedded in the atelocollagen remarkably suppressed human tumor growth over a 35–42 day period.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Biomaterials

Antigenic telopeptides attached to both ends of the collagen molecules are eliminated by pepsin treatment, resulting in the formation of atelocollagen.25

Oligonucleotides

The oligonucleotides (ODNs) used were phosphorothioate ones. The sequences for ODC antisense ODNs (targeting the human ODC translation initiation region, 319–338, NM 002539) and scrambled ODNs were 5′ TCA TGA TTT CTT GAT GTT CC 3′ and 5′ CCT TGT AGT TCT TTA GTA CT 3′, respectively.27

Treatment of Cells in Culture With Antisense Oligonucleotides

MKN45 (gastric cancer) and COLO201 (colon cancer) cells were cultured in RPMI and RD (rhabdomyosarcoma) cells in DMEM containing 10% fetal calf serum (Health Science Research Resource Bank, Osaka, Japan). Five × 105 cells/500 μL media per well were treated with 10 μM antisense ODNs in the atelocollagen (0.018%), 10 μM scrambled ODNs in the atelocollagen, 10 μM scrambled ODNs, 10 μM antisense ODNs, 0.018% atelocollagen, and 10 μM antisense ODNs in the atelocollagen (0.018%) + 20 μM putrescine. At 2, 4, and 6 days the cell number was evaluated using both the MTT assay (Wako, Tokyo) and a cell-counting method using a hemocytometer under a microscope in triplicate. Similar results were obtained. Thus, we show the results obtained by the cell-counting method using a hemocytometer in the Results. The efficiency of transfection was evaluated using 10 μM FITC-labeled ODC antisense ODNs in the atelocollagen (0.018%). The percentage of positive cells at 8 and 20 hours in MKN45, COLO201, and RD cells was (47.0 ± 8.2, 59.6 ± 3.7), (58.3 ± 8.6, 62.0 ± 5.8), and (25.4 ± 4.6, 38.7 ± 4.6), respectively.

Measurement of ODC Activities

ODC activities were determined in triplicate using DL-[l-14C]-ornithine (Amersham Pharmacia Biotech, Buckinghamshire, UK) as a substrate.12 The protein concentration was measured using a Bradford assay kit (Bio-Rad, Tokyo).

Measurement of Polyamine Levels by High-Performance Liquid Chromatography

The contents of polyamines were measured using high-performance liquid chromatography28 and are expressed as percent compared with control.

Measurement of Antitumor Effects of ODC Antisense ODNs in Nude Mouse Tumor Models

This study was approved by a DNA committee, Faculty of Medicine, the University of Tokyo, and all procedures involving animal care were in accord with institutional guidelines in compliance with national laws. Male BALB/c Slc-nu nude mice (4–5 weeks old, 16–20 g; SLC, Hamamatsu City, Japan) were anesthetized with ethylether and inoculated subcutaneously with 5 × 107 MKN45, COLO201, and RD cells on the back. After about 7 days (6–8 days) when palpable tumors reached approximately 200 mg the treatment was started. The weights were calculated by the formula l × w2/2 [length (l), and width (w)]. One hundred μL of saline solution containing ODC antisense ODNs or scrambled ODNs (500 μg) embedded in the atelocollagen (1.8%) was directly injected into tumor or intraperitoneally (i.p.) or intramuscularly (i.m.) on the back of mice.

To clarify the difference of efficiency of liposome and atelocollagen, we compared the effect of treatment of liposome (Transfast) + AS ODC via local injection and that of atelocollagen + AS ODC via local injection on RD tumor growth. The liposome called Transfast was purchased from Promega (Madison, WI). It is composed of ±N,N'bis(2-hydroxyethyl)-N-methyl-N-[2,3-di (tetradecanoyloxy) propyl] ammonium iodido and neutral lipid (DOPE).

Histological Study

At 35 (MKN45 and COLO201) and 42 (RD) days after treatment, tumor tissues were removed and stained with hematoxylin and eosin or with the Azan method. FITC-labeled ODC antisense ODNs with and without atelocollagen were administered i.p. in RD tumor-bearing mice and the intensity of fluorescence was compared.

Statistical Analysis

The significance was determined by analysis of variance (ANOVA) followed by the Fisher PSLD test or by Scheffe test (indicated with an asterisk). All P-values were 2-sided.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Effects of ODC Antisense ODNs on Cell Growth In Vitro

MKN45, COLO201, and RD cells with the highest ODC activities were selected. In dose-response experiments (0–25 μM), 10 μM antisense ODNs in the atelocollagen (0.018%) were the most effective in RD cell growth in vitro (data not shown). Antisense ODNs in the atelocollagen markedly suppressed MKN45, COLO201, and RD cell growth (Fig. 1), 83.8% (P < .0001), 86.5% (P < .0001), and 95.6% (P < .0001), respectively. Scrambled ODNs had no significant inhibitory effect on MKN45 and COLO201 cell growth (P = .6281 and P = .4137, respectively). Atelocollagen inhibited MKN45, COLO201, and RD cell growth 26.8% (P = .0027), 35.4% (P = .0001), and 54.2% (P < .0001), respectively. Scrambled ODNs in the atelocollagen also inhibited cell growth 32.3% (P = .0006), 38.0% (P < .0001), and 59.9% (P < .0001), respectively, which was considered to be the effect of atelocollagen. Antisense ODNs also inhibited cell growth 56.1% (P < .0001), 29.7% (P = .0006), and 69.1% (P < .0001), respectively. The effect was smaller than antisense ODNs in the atelocollagen (P = .0140, P < .0001, and P = .0011). The specificity of growth inhibitory effect of antisense ODNs in the atelocollagen was shown by the following results. Twenty μM putrescine completely blocked the inhibitory effect induced by antisense ODNs in the atelocollagen on MKN45 cell growth (P = .3014) (Fig. 1A), and rescued the inhibitory effect on COLO201 and RD cell growth up to 87%, and 78%, respectively (Fig. 1B,C). Although the inhibitory effect of atelocollagen was unexpected, it seems to be beneficial for ODC antisense therapy with atelocollagen.

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Figure 1. Effects of ornithine decarboxylase (ODC) antisense oligonucleotides (ODNs) on cell growth. Time course of effects of 10 μM antisense ODNs embedded in the atelocollagen (0.018%) (σ), atelocollagen (0.018%) (λ), 10 μM scrambled ODNs (Δ), 10 μM scrambled ODNs in the atelocollagen (υ), 10 μM antisense ODNs (□), 20 μM putrescine + 10 μM antisense ODNs in the atelocollagen (0.018%) (o), and control (ν) on 3 cell growths was studied in vitro. Cell number was determined by counting cells using a hemocytometer under microscope. (A) MKN45; (B) COLO201; (C) RD.

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Cell Viability by Trypan Blue Method

The viability of MKN45, COLO201, and RD cells at 6 days was 56.0 ± 5.3% (P < .0001), 57.8 ± 9.2% (P < .0001), and 42.1 ± 10.3% (P < .0001), respectively. Cell death was, at least in part, responsible for the growth inhibition induced by ODC antisense ODNs in the atelocollagen.

Effects of ODC Antisense ODNs on ODC Activities and Polyamine Levels

At 6 days antisense ODNs in the atelocollagen suppressed ODC activities in MKN45, COLO201, and RD cells, 93.3% (P < .0001), 90.5% (P < .0001), and 86.4% (P < .0001), respectively (Fig. 2). Although scrambled ODNs had no significant inhibitory effect on ODC activities (P = .3365, P = .8919, and P = .2941), atelocollagen inhibited ODC activities in 3 cells, 41.3% (P < .0001), 37.2% (P = .0003), and 54.5% (P < .0001), respectively. Scrambled ODNs in the atelocollagen also inhibited ODC activities, 48.0% (P < .0001), 34.0% (P = .0006), and 54.5% (P < .0001), respectively, which was considered to be the effect of atelocollagen. Antisense ODNs also inhibited ODC activities, 62.7% (P < .0001), 54.3% (P < .0001), and 50.0% (P < .0001), respectively. But the effect of antisense ODNs on ODC activities was less than that of antisense ODNs in the atelocollagen (P = .0004, 0.0017, and 0.0007, respectively). Suppression of ODC activities was almost completely blocked by 20μM putrescine (Fig. 2) (P = .8451, P = .0731, and P = .0468, respectively), indicating the specificity of ODC antisense ODNs in the atelocollagen. At 6 days, antisense ODNs in the atelocollagen suppressed putrescine and spermidine contents in MKN45, COLO201, and RD cells [95.3 ± 4.1% (P < .0001), 53.1 ± 9.3% (P = .0008) (n = 3)], [88.7 ± 9.1% (P < .0001), 73.3 ± 13.3% (P = .0002) (n = 3)], and [76.2 ± 2.2% (P < .0001), 45.6 ± 16.7% (P = .0036) (n = 3)], respectively. Spermine contents remained unchanged or slightly decreased.

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Figure 2. Effects of ornithine decarboxylase (ODC) antisense oligonucleotides (ODNs) on ODC activities in vitro. Time course of effects of 10 μM antisense ODNs (AS) in the atelocollagen (0.018%) (σ), atelocollagen (0.018%) (λ), 10 μM scrambled ODNs (Δ), 10 μM scrambled ODNs in the atelocollagen (υ), 10 μM antisense ODNs (□), 20 μM putrescine + 10 μM antisense ODNs in the atelocollagen (0.018%) (o), and control (ν) on ODC activities in 3 cells in vitro was studied. ODC activities at 2, 4, and 6 days were measured. (A) MKN45; (B) COLO201; (C) RD.

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Antitumor Effects of ODC Antisense ODNs In Vivo

Antisense ODNs in the atelocollagen remarkably suppressed tumor growth over a 35–42 day period (Fig. 3). At 35 days MKN45 tumor growth with antisense ODNs in the atelocollagen was almost completely suppressed (Fig. 3A). The size was 0.2% (P < .001*) compared with nontreated control. Tumor growth treated with the atelocollagen and scrambled ODNs + atelocollagen was also significantly suppressed and the size was 34.9% (P < .0001*) and 41.0% (P < .0001*), respectively. This effect was considered to be the effect of atelocollagen. At 35 days COLO201 tumor growth with antisense ODNs in the atelocollagen was remarkably suppressed and the size was 3.7% (P < .0001*) (Fig. 3B). Atelocollagen suppressed COLO201 tumor growth and the size was 9.5% (P < .0001*). At 42 days RD tumor with the antisense ODNs in the atelocollagen was remarkably suppressed and the size was 13.7% (P < .0001*) (Fig. 3C). Atelocollagen suppressed RD tumor and the size was 44.2% (P < .0001*). The effect was less than that with antisense ODNs in the atelocollagen (P < .0001*). We next compared the effect of antisense ODNs in the atelocollagen with antisense ODNs in liposome (TransFast). Antisense ODNs in liposome suppressed RD tumor 38.5% (P < .0001*), but the effect was smaller than that of antisense ODNs in the atelocollagen (P < .0001*) (Fig. 3C).

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Figure 3. Effects of ornithine decarboxylase (ODC) antisense oligonucleotides (ODNs) on the growth of tumor in nude mice. Tumor volume was determined after intratumoral administration of antisense ODNs in the atelocollagen (n = 10) (λ), scrambled ODNs in the atelocollagen (n = 10) (□), and atelocollagen (n = 10) (ν). The effects of intraperitoneal (n = 5) (Δ) and intramuscular (o) single administration of antisense ODNs in the atelocollagen (n = 5) and of intratumoral administration of antisense ODNs in liposome (TransFast) (n = 5) (σ) on RD tumor growth was also evaluated. Control is shown as (ν). The effects of intraperitoneal single administration of antisense ODNs in the atelocollagen on MKN45 (n = 20) (Δ) and COLO201 (n = 20) (Δ) tumor growth were also evaluated. N indicates number of mice treated. (A) MKN45; (B) COLO201; (C) RD.

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Comparison of Effects of Antisense ODNs on Tumor Growth via Different Administration Routes

Administration of antisense ODNs in the atelocollagen via i.m. and i.p. injection remarkably suppressed RD tumor (Fig. 3C). Intraperitoneal administration of antisense ODNs in the atelocollagen was the most effective on RD tumor suppression. The size of tumor 42 days after administration of antisense ODNs in the atelocollagen via i.m., i.p., and intratumoral (i.t.) injection was 19.9% (P < .0001*), 3.5% (P < .0001*), and 13.7% (P < .0001*), respectively (Fig. 3C). At 35 days after i.p. injection the growth of MKN45 and COLO201 was almost completely suppressed. The size was 0.7% (P < .0001*) and 3.5% (P < .0001*), respectively (Fig. 3A,B).

Effects of ODC Antisense ODNs on ODC Activities and Polyamine Levels In Vivo

Intratumoral administration of ODC antisense ODNs in the atelocollagen significantly suppressed ODC activities in MKN45, COLO201, and RD tumors 87.8% (n = 10, P < .0001*), 64.9% (n = 8, P < .0001*), 71.3% (n = 6, P < .0001*), respectively (Fig. 4). Atelocollagen (n = 10) inhibited ODC activities in 3 tumors (Fig. 4A-C), but the effect was less than that of antisense ODNs in the atelocollagen (P < .0001*, P = .0006*, and P < .0001*, respectively). Scrambled ODNs in the atelocollagen (n = 10) slightly inhibited ODC activities in MKN45 cells (Fig. 4A), but its effect was less than antisense ODNs in the atelocollagen (P < .0001*, n = 10). ODC antisense ODNs in liposome (TransFast) (n = 5) also inhibited ODC activities in RD cells (Fig. 4C), but its effect was less than antisense ODNs in the atelocollagen (P < .0001*) (n = 10). Antisense ODNs in the atelocollagen administered i.p. suppressed ODC activities in 3 tumors, 89.9% (n = 20, P < .0001*), 81.9% (n = 20, P < .0001*), and 85.6% (n = 5, P < .0001*), respectively (Fig. 4A-C). Antisense ODNs in the atelocollagen administered i.m. significantly suppressed ODC activities 61.2% in RD tumor (n = 3, P = .0003) (Fig. 4C).

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Figure 4. Effects of ornithine decarboxylase (ODC) antisense oligonucleotides (ODNs) on ODC activities in tumors. Effects of a single intratumoral administration of antisense ODNs (AS) in the atelocollagen, atelocollagen, and scrambled ODNs (SCR) in the atelocollagen on ODC activities in MKN45, COLO201, and RD tumors was studied. Effects of intratumoral (local), intramuscular, and intraperitoneal single administration of ODNs in the atelocollagen on ODC activities were also measured. At 35 (MKN45, COLO201) and 42 days (RD) ODC activities were determined. (A) MKN45; (B) COLO201; (C) RD.

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Intratumoral administration of antisense ODNs in the atelocollagen remarkably suppressed putrescine contents, 89.1 ± 18.0% (n = 3, P < .0001), 71.6 ± 8.8% (n = 3, P = .0038), and 83.7 ± 19.8% (n = 3, P < .0001) in MKN45, COLO201, and RD tumors, respectively. Spermidine contents were also suppressed 67.2 ± 22.4% (n = 3, P < .0001), 49.4 ± 24.7% (n = 3, P = .0007), and 73.7 ± 1.8% (n = 3, P < .0001), respectively. The spermine contents were not significantly affected in 3 tumors (P = .4473, P = .6755, and P = .7220, respectively). Intraperitoneal administration of antisense ODNs in the atelocollagen remarkably suppressed putrescine and spermidine contents [92 ± 13.2% (P < .0001), and 60 ± 14.7% (P = .0003)], [81 ± 22.6% (P = .0020) and 71.8 ± 8.2% (P < .0001)], [83.7 ± 14.8% (P < .0001) and 73.7 ± 4.2% (P < .0001)] in MKN45, COLO201, and RD tumors (n = 3), respectively.

Histologic Analysis

Effects of a single i.t. administration of antisense ODNs in the atelocollagen on tumor tissue and other organs were analyzed histologically under a microscope. At 35 (MKN45 and COLO201) and 42 (RD) days after treatment tumor tissues were removed and stained with hematoxylin and eosin. Collagen was stained with the Azan method. Typical examples in MKN45, COLO201, and RD tumor tissues are shown in Figure 5. Histological examination (H&E stain) showed marked necrosis, surrounded by a small number of living cancer cells, indicating that administration of antisense ODNs in the atelocollagen induced necrosis. Tumor tissues stained by the Azan method showed a marked proliferation of fibrous tissue and collagenous fiber. Control tumor tissues showed a marked proliferation of cancer cells (H&E), surrounded by a slight or modest proliferation of fibrous tissue. We purified genomic DNAs from tumor tissues treated with antisense ODNs in the atelocollagen. A DNA ladder was not detected (data not shown). In other organs examined no abnormal macroscopic and histologic changes were observed (not shown), indicating that antisense ODNs in the atelocollagen gave no apparent toxicity in other organs.

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Figure 5. Histological analysis of effects of ornithine decarboxylase (ODC) antisense oligonucleotides (ODNs) in the atelocollagen on tumor tissue. Effects of a single intratumoral administration of antisense ODNs (AS) in the atelocollagen on tumor tissue were analyzed histologically under a light microscope. At 35 (MKN45 and COLO201) and 42 (RD) days after treatment, treated tumor tissues and control tumor tissues were removed, fixed, and stained with hematoxylin and eosin (H&E). Collagen was stained with the Azan method. Magnification, ×200.

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Detection of Fluorescence in Tumor Administered FITC-Labeled ODC Antisense ODNs in the Atelocollagen

We next examined whether antisense ODNs in the atelocollagen were taken up into tumor after i.p. injection. FITC-labeled antisense ODNs (500 μg) in the atelocollagen (1.8%) or FITC-labeled antisense ODNs were administered i.p. in RD tumor (about 200 mg)-bearing nude mice. After 3 days the tumor was removed and observed under a fluorescent microscope. Fluorescence was clearly detected in the tumor (Fig. 6). The intensity of the fluorescence was quantified. The intensity in tumor administered with FITC-labeled antisense ODNs in the atelocollagen was 3.1 ± 0.6-fold (P < .005) compared with that in tumor administered with FITC-labeled antisense ODNs. The data indicate a pivotal role of atelocollagen in protecting ODNs from nuclease attack.

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Figure 6. Detection of fluorescence in tumors after intraperitoneal administration of FITC-labeled ornithine decarboxylase (ODC) antisense oligonucleotides (ODNs). FITC-labeled antisense ODNs (AS) (500 μg) with and without atelocollagen (1.8%) were administered intraperitoneally in RD tumor (around 200 mg in size)-bearing nude mice. On 3 day after administration tumors were removed and sections were cut. Fluorescence was detected using a fluorescent microscope. The intensity of fluorescence with (FITC-AS + atelocollagen) and without atelocollagen (FITC-AS) was compared. Corresponding hematoxylin and eosin (H&E)-stained photos are shown in the right panel. Magnification, ×200.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

We have shown that administration of ODC antisense ODNs in the atelocollagen markedly suppressed the growth of human stomach, colon, and rhabdomyosarcoma tumor in vitro and in vivo. A striking point in this study is that a single administration of ODC antisense ODNs in the atelocollagen via i.t., i.p. and i.m. routes had remarkable tumor suppressive effects for at least 35–42 days. Atelocollagen played a pivotal role in achieving this effect by preventing nuclease access to ODNs, as we observed in our previous study in a case of plasmid DNA in the atelocollagen.25, 26 A single injection of ODC antisense ODNs embedded in the atelocollagen allowed continuous suppression of tumor growth, ODC activities, and polyamine levels for at least 35–42 days without additional administration of ODC antisense ODNs. This indicates that ODC antisense ODNs embedded in the atelocollagen remained intact for 35–42 days in vivo and seemed to be released gradually. Atelocollagen is useful to avoid rapid degradation of ODC antisense ODNs in vivo. Inhibition of cell and tumor growth by ODC antisense ODNs in the atelocollagen was associated with decreased ODC activities and polyamine levels, especially with decreased levels of putrescine in cells or tumor. The results are consistent with those shown in difluoromethylornithine (synthetic inhibitor of ODC)-induced tumor suppression and cell death.29–32

One of the major limitations for the therapeutic use of antisense ODNs is the problem of delivery.21, 22, 33–35 Many classes of compounds have been used as delivery vehicles including cationic liposomes.33, 35 Relatively high cytotoxic properties of cationic liposomes may restrict their use in in vivo experiments. We compared our system with the commercially available cationic liposome (TransFast). ODC antisense ODNs in TransFast suppressed RD tumor growth, but it was less effective than our system. There are reports that describe in vivo efficacy with antisense ODNs targeted against various genes such as c-raf-1 kinase, c-myc, and protein kinase A,21, 22 but repeated administration of antisense ODNs is necessary to obtain sufficient tumor-suppressive effects. Daily i.v. injection of antisense phosphorothioate c-raf-1 kinase oligonucleotides (up to 6 mg/kg) was necessary to inhibit tumor growth efficiently,36 and clinical responses were observed only in 3 of 17 ovarian cancer.37 Antisense ODNs directed against PKC-alpha has been tested in some human tumor cell lines grown in athymic mice.38 Administration of ODNs, once a day for 14 days, was necessary to obtain a noticeable tumor suppressive effects with a 50% inhibitory dose between 60 and 600 μg/kg per day. This ODN entered a Phase I clinical trial.39, 40 The effect of antisense ODNs targeted to the c-myc oncogene was tested on the growth of human melanoma xenografts.41 One mg per mouse per day, i.v., daily for 8-day antisense treatment inhibited tumor growth. A single-injection (50 mg/kg) protein kinase A-directed antisense ODN treatment inhibited colon tumor growth.42 Several Phase I/II trials using antisense ODNs have been reported, but the effect was modest.43–47

Bcl-2-specific antisense oligonucleotides have shown broad anticancer activities in preclinical models and are currently in several Phase III trials.48, 49

Our system also seems to be more applicable for clinical use as a single administration of ODC antisense ODNs-induced maximum tumor-suppressive effects over a period of 35–42 days without any noticeable toxic effects in other organs.

In summary, ODC antisense ODN therapy using atelocollagen as a carrier had a remarkable growth-suppressive effect on human stomach, colon, and rhabdomyosarcoma tumor. As a single administration of ODC antisense ODNs had a continuous tumor suppressive effect and various human cancers express higher levels of ODC, this novel method of targeting against ODC expression may be of considerable value for clinical applications.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES